Clinicians design antiretroviral therapy to prevent HIV-1 replication and resistance, and researchers study antiretroviral concentrations to understand the pharmacokinetics of these drugs. Because drug efficacy and toxicity varies widely between patients receiving the same antiretroviral therapy, there is interest in monitoring individual patient concentrations of antiretroviral drugs. Good science and effective medical care demand inexpensive validated methods with high throughput that are capable of simultaneously analyzing multiple antiretroviral drugs in various matrices. Currently, protease inhibitors, non-nucleoside reverse transcriptase inhibitors, and nucleoside reverse transcriptase inhibitors are used to treat HIV-1 infection. This review summarizes published methods for the quantitation of nucleoside reverse transcriptase inhibitors and their metabolites in different matrices using immunoassays, ultraviolet absorption, and mass spectrometry.